Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression.

Housekeeping genes are widely used as internal controls for gene expression normalization for western blotting, northern blotting, RT-PCR, etc. They are generally thought to be expressed in all cells of the organism at similar levels because it is assumed that these genes are required for the maintenance of basic cellular function as constitutive genes. However, real-time RT-PCR experiments revealed that their expression may vary depending on the developmental stage, type of tissue examined, experimental condition, and so on. To date, no histological data on their expression are available for embryonic development. In the present study, we compared the histological expression profile of two commonly used housekeeping genes, GAPDH and beta-actin, in the developing chicken embryo by using section and whole mount in situ hybridization supplemented by RT-PCR. Our results show that neither GAPDH mRNA nor beta-actin mRNA is expressed in all cell types or tissues at high levels. Strikingly, expression levels are very low in some organs. Moreover, the two genes show partially complementary expression patterns in the liver, the vascular system and the digestive tract. For example, GAPDH is more strongly expressed in the liver than beta-actin, but at lower levels in the arteries. Vice versa, beta-actin is more strongly expressed in the gizzard than GAPDH, but it is almost absent from cardiac muscle cells. Researchers should consider these histological results when using GAPGD and beta-actin for gene expression normalization in their experiments.